Microscope diagnostic characterization of hematologic neoplasms is increasingly dependent on immunophenotypic analysis, often performed by flow cytometry. Immunophenotyping by flow cytometry provides objective data that is necessary for the subclassification of leukemia and lymphoma, and that can often definitely establish or exclude a diagnosis of malignancy in seemingly borderline cases. Immunophenotyping of peripheral blood leukocyte subsets is also an important laboratory indicator for patients infected with the human immunodeficiency virus (HIV) that causes the acquired immunodeficiency syndrome (AIDS). Flow cytometers are used routinely to provide dual antibody profiles using patient blood and other specimens such as tissue biopsies. Each set of dual measurement requires a separate tube containing cells treated with two antibodies. Each tube is independently run on the flow cytometer and the results of all measurements are combined on a report. Although it is possible to measure as many as four different antibodies at the same time on each cell with a flow cytometer, this is technically difficult and expensive. Therefore, multiple aliquots of dual antibodies are more commonly utilized.
Drawbacks of flow cytometry include relatively large specimen size requirements and the inability to microscopically examine the cells being studied. Furthermore, in order to normally accomplish multi-parameter immunophenotyping (greater than four immunofluorescent antibodies per specimen), flow cytometric analysis requires that separate subsets of a single specimen be manually or robotically placed within the flow cytometer and analyzed separately.
Until now, alternatives to flow cytometry have been few, including only immunohistochemical methods that suffer from a lack of automation and objectivity, and that generally compromise the number of antigens that can be analyzed.
A device, referred to as a laser scanning cytometer, available from CompuCyte Corp. (under the trademark "LSC", as described in U.S. Pat. No. 5,072,382, the disclosure of which is incorporated herein by reference thereto), has been recently developed which provides an alternative to flow cytometric immunophenotyping, and offers several unique advantages. Similar to flow cytometry, laser scanning cytometry provides objective automated, multi-color analyses of hematologic cell suspensions, stained by using conventional immunofluorescent techniques. However, laser scanning cytometry also allows for the visual examination of individual cells both during and after analysis, and makes possible the analysis of very small diagnostic samples, such as those obtained via fine needle aspiration biopsy. This is true primarily because for laser scanning cytometry, the specimens are analyzed on a static surface (such as a glass microscope slide) rather than within a fluid stream as with flow cytometry. An added advantage and the principal idea behind the present invention is that for laser scanning cytometry true multi-parameter immunophenotyping of a single specimen (or even multiple specimens) is possible using only a single glass microscope slide or other vehicle to load the specimen into the laser scanning cytometer.
Preparation of a specimen for immunophenotypic analysis is performed by reacting aliquoted subsets of that specimen (typically constituting a suspension of purified hemopoietic or lymphoreticular cells) with various mixtures of fluorochrome-labelled antibodies. After this reaction, the aliquoted subsets are washed and analyzed individually via flow cytometry or laser scanning cytometry. Typically each aliquot is reacted simultaneously with a mixture of up to four different antibodies labelled with different fluorochromes. This is possible because of differences in the spectral characteristics of the fluorochromes utilized and because the instrumentation of flow cytometer or the laser scanning cytometer is designed to simultaneously but discretely assay emitted light from such fluorochromes. Because of the cost of producing different antibodies labeled with various fluorochromes and the cost and complexity of instrumentation necessary for the assay, individual aliquoted subsets of a single specimen are usually simultaneously reacted with only two or three different antibodies. However, modern immunophenotypic analysis usually requires assessment of multiple (usually 5 to 15) different antigens, and it is often clinically necessary or desirable to assess a single particular antigen in the context of multiple different other antigens. Therefore, specimens are presently aliquoted and reacted with multiple mixtures of various fluorochrome-labelled antibodies to is assess the expression of the corresponding antigens and their relationships with one another.
For testing of the multiple mixtures, at least three manufacturers sell slides with multiple compartments or devices which can create multiple compartments. These do not, however, provide means for generating an assay and they also require separate aliquot samples for each of the compartments or chambers.